WO2014106720A1

WO2014106720A1 - Peptide for use in the treatment of motor neuronopathies

Info

Publication number: WO2014106720A1
Application number: PCT/FR2014/050002
Authority: WO
Inventors: Suzie LEFEBVRE; Alphonse Garcia
Original assignee: Centre National De La Recherche Scientifique - Cnrs -; Institut Pasteur
Priority date: 2013-01-04
Filing date: 2014-01-03
Publication date: 2014-07-10
Also published as: FR3000746A1

Abstract

The invention relates to a peptide comprising a penetration sequence (a); and a sequence (b) comprising the protein pattern shown in SEQ ID NO. I, in particular for use in the treatment of a motor neuronopathy.

Description

Peptide for use in the treatment of motor neuronopathies

The invention relates to a peptide for its use in the treatment of motor neuronopathy.

Infant spinal muscular atrophy (or SMA, Spinal Muscular Atrophy) is an autosomal recessive neuromuscular disease. It is characterized by degeneration of the alpha motor neurons of the anterior horn of the spinal cord leading to muscle atrophy and leading to paralysis. This degeneration of alpha motor neurons therefore substantially compromises the vital prognosis of patients. In the healthy subject, these neurons transmit messages from the brain to the muscles, causing the contraction of the latter. In the absence of such stimulation, the muscles atrophy. Thereafter, in addition to widespread weakness and atrophy of the muscles, particularly those of the trunk, upper arms and thighs, these disorders can be accompanied by serious respiratory problems.

There is a strong correlation between the age of onset of symptoms and the severity of the disease, so that the earlier the disease starts and the less likely it is to be life-threatening. It is according to this criterion that this disease has been classified into 3 clinical types as follows:

type I (Wernig-Hoffman's disease), characterized by an early onset, usually before 6 months, in which the sitting position is never acquired and the course is extremely serious. The life expectancy of affected children rarely exceeds three years and is often limited to a few months,

the type II or intermediate form of Wernig-Hoffman's disease that usually appears between the age of six months and three years, in which the sitting station is acquired, but the walk is never. This clinical type is often associated with frequent respiratory infections that can complicate the cause of this condition and decrease life expectancy.

Type III or Kugelberg-Welander disease, which usually appears around 3-4 years of age and sometimes up to 21 years of age and in which walking is acquired with more or less pronounced gait disturbances depending on the severity which is very variable from one patient to another. This type of illness in general does not compromise life expectancy.

All forms of spinal muscular atrophy are accompanied by progressive muscle weakness and atrophy, as a result of the degeneration of the neurons of the anterior horn of the spinal cord. The ADM is currently one of the most common causes of child mortality. It affects girls and boys alike in all regions of the world with a prevalence of between 1/6000 and 1/10000.

The gene involved in spinal infantile amyotrophy was located on chromosome 5 in the q12-q13 position, regardless of the clinical type presented. This gene encodes the motor neuron survival protein (SMN). This protein is localized in the cytoplasm and nucleus, where it accumulates in so-called elm areas called Cajal bodies or Cajal bodies (CBs). A defect of this accumulation is described in the SMA.

It is recognized that SMA is related to inactivation of the SMN gene. More specifically, two genes encoding the SMN protein: SMN1 and SMN2, the two genes being transcribed, were highlighted. The analysis of their promoters has shown that these elements are almost identical both in their sequence and in their activity. Also, the SMN2 gene encodes the same SMN protein but in lesser amounts. It has moreover been found that the inactivation of SMN1 can be partially mitigated by the expression of the quasi-identical SMN2 gene.

Patients with ADS currently have medical or paramedical care to ensure the best possible quality of life, such as:

motor physiotherapy and hydrotherapy, which consist in helping the patient to build up his body diagram;

- Respiratory physiotherapy, which relieves patients of bronchial congestion, thus reducing the risk of respiratory diseases; or Orthopedic care to prevent skeletal and joint deformities.

However, this care is only intended to relieve symptoms and prevent complications of the disease. They do not heal the pathology.

On the other hand, amyotrophic lateral sclerosis (ALS) or Charcot's disease is a neurodegenerative disease mainly affecting adults, characterized by weakening and then paralysis of the muscles of the legs and arms, the respiratory muscles, and the muscles of the muscles. swallowing and speech. The prevalence of this disease is estimated at 1 patient out of 25,000 people.

This disease also involves the degeneration of motor neurons, particularly those of the cerebral cortex and the anterior horn of the spinal cord. This degeneration leads to a destruction of the pyramidal tract. ALS can occur in two main forms: the "spinal" form and the "bulbar" form. The spinal form is due to the degeneration of the motoneurons located in the spinal cord while the bulbar form corresponds to the degeneration of the motoneurons of the medulla oblongata. These two forms may succeed or develop simultaneously, the disease progressing almost always towards a complete form (spinal and bulbar).

Patients suffering from ALS have medical care aimed not at the recovery of motor functions but at the maintenance of the remaining functions, notably through physiotherapy and orthopedic rehabilitation. In addition, a therapeutic strategy based on Riluzole (marketed under the name Rilutek®) has recently been proposed. But this strategy only allows to delay the evolution of the effective pathology and prolongs the phase of the disease during which the patient is autonomous. Also, this compound improves the prognosis of patients by only 30, which remains unsatisfactory. There is currently no effective treatment for the onset and / or development of ALS. There is no treatment to effectively stop the progression of motor neuronopathies, especially to stop the degeneration of motor neurons. There is therefore a long-standing need for an effective therapeutic strategy against motor neuronopathies, in particular the motor neuronopathies of the spinal amyotrophic infantile type and amyotrophic lateral sclerosis.

The inventors have now identified new partners of the SMN protein, in particular protein-phosphatase ΡΡΙγ, which is a protein regulating protein interactions.

They showed on the one hand that ΡΡΙγ plays a role in the formation of the SMN complex and its localization to CBs, and on the other hand that the ΡΡΙγ proteins interact with the Gem-associated protein 8 also called Géminé 8, which is a SMN complex protein. Indeed, the ubiquitous SMN complex is formed of the Gem-associated proteins 2 to 8 (Géminé 2-8) and unrip.

After long and tedious work, the inventors have developed a peptide mimicking the site of interaction between Géminé 8 and ΡΡΙγ. They highlighted that this peptide is a promising strategy to restore the deficit of SMN protein and thus treat motor neuronopathies.

Peptide of the invention

The invention therefore relates to a peptide comprising:

a sequence (a) of penetration;

a sequence (b) comprising the protein unit shown in SEQ ID NO: 1 (HHVAWJ.

Typically, the peptide of the invention is an isolated peptide. Typically, sequences (a) and (b) are amino acid sequences.

By "penetration sequence" or "penetration shuttle" or "cellular shuttle" is meant an amino acid sequence having the capacity to induce the cellular penetration of a peptide comprising said penetration sequence within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or else 100% of the cells constituting a given population of cell culture . Also, the presence of a sequence of penetration into the structure of a peptide allows said peptide to enter cells after contacting under appropriate conditions, well known to those skilled in the art. This cellular penetration consists of the passage of said peptide from the external environment into the intracellular medium, including the cytoplasm, under conditions much higher than those encountered during passive diffusion. Typically, said penetration sequence (a) is a sequence comprising cationic amino acids.

Preferably, this penetration sequence (a) is biologically inactive.

By "biologically inactive penetration sequence" is meant a sequence which induces no significant biological effect. In addition, a biologically inactive penetration sequence is a sequence that is not capable of internalizing protein complexes.

Typically, a biologically inactive penetration sequence, taken alone, is not likely to have a beneficial effect in therapy, particularly in the treatment of motor neuronopathy.

In the context of the invention, when a biologically inactive penetration sequence (a) is coupled to a sequence (b) as represented in SEQ ID No. 1, the peptide of the invention thus constituted acquires:

the ability to internalize a protein complex, typically horseradish peroxidase (HRP);

a biological effect, especially in therapy.

Preferably, said penetration sequence (a) is an amino acid sequence comprising between 6 and 8 cationic amino acids.

Typically, said penetration sequence (a) is selected from the group consisting of the following amino acid sequences: SEQ ID NO: 2 (VKKKKIKREIKI);

SEQ ID No. 5 (RQKRLIRQKRLIRQKRLI);

SEQ ID NO: 6 (RRRRRRRSRGRRRRTY);

SEQ ID No. 7 (PRRPGPTRKHYQPYA);

SEQ ID NO: 8 (VKKKKIKREIKIPRRPGPTRKH YQP Y A);

SEQ ID No. 9 (FRGRSRFRGRSR);

SEQ ID NO: 10 (VKKKKIKREIKIFRGRSRFRGRSR);

SEQ ID No. 11 (VKKKKIKREIKIARGRSRFRGRSR);

SEQ ID NO: 12 (VKKKKIKREIKIARGRSRARGRSR);

SEQ ID No. 14 (RQIKrWFQNRRMKWKK);

SEQ ID No. 15 (GWTNLS AGYLLGKINLKALA ALAKKIL);

SEQ ID No. 16 (GALFLGFLGGAAGSTMGAWSQPKSKRKV);

SEQ ID No. 18 (DAATATRGRSAARPTERPRAPARSASRPRRPVE);

SEQ ID No. 19 (GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR);

- SEQ ID ^No. 20 (KETWWETWWTEWSQPKKKRKV);

SEQ ID NO: 21 (KLALKLALKALKAALKLA);

SEQ ID No. 22 (GALFLGWLGAAGSTMGAPKKKRKV);

SEQ ID No. 23 (RRWWRRWRR);

SEQ ID NO: 24 (RRRRRRRRRR);

SEQ ID No. 25 (RHSRIGIIQQRRTRNG); and

SEQ ID NO: 26 (KGVIFYLRDK).

More preferably, said penetration sequence (a) is an amino acid sequence comprising between 6 and 8 amino acids chosen from arginine and lysine residues.

Even more preferably, said penetration sequence (a) has an amino acid sequence as represented by SEQ ID No. 2 (VKKKKIKREIKI). This sequence (a) is also called "DPT-shl" or "shuttle DPT-shl".

In a preferred embodiment, said sequence (b) has an amino acid sequence as represented by SEQ ID NO: 3 (AYPQSFYDHHVAWQDYPCS). In a preferred embodiment, the peptide of the invention consists of the sequence represented by SEQ ID NO: 4 (VKKKKIKREIKIAYPQSFYDHHVAWQDYPCS). This peptide is also referred to as "DPT-S1". Alternatively, the peptide of the invention consists of a sequence having at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% identity with the sequence SEQ ID No. 4. Alternatively, the peptide of the invention consists of the sequence SEQ ID No. 4, for which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids have been substituted.

Alternatively, the peptide of the invention is a fragment of the sequence SEQ ID No. 4. Preferably, said fragment comprises the sequence SEQ ID No. 1.

Typically, this fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or 30 amino acids of the sequence represented by SEQ ID NO: 4.

By "percent identity" between two amino acid sequences in the sense of the present invention, it is meant to designate a percentage of identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistics and the differences between the two sequences being randomly distributed over their entire length. By "best alignment" or "optimal alignment" is meant the alignment for which the percentage of identity determined as hereinafter is the highest. Sequence comparisons between two amino acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being performed by segment or by "comparison window" to identify and compare the local regions of sequence similarity. . The optimal alignment of the sequences for comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970) ), using the similarity search method of Pearson and Lipman (1988), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).

In another embodiment, said penetration sequence (a) is chosen from:

the amino acid sequence shown in SEQ ID NO: 27 (CRRWWRRWRR), whose cysteine in position 1 forms a disulfide bridge with cysteine at position 18 of the sequence SEQ ID NO: 3; and

the amino acid sequence represented in SEQ ID No. 28 (CRRRRRRRRRR), whose cysteine in position 1 forms a disulfide bridge with the cysteine at position 18 of the sequence SEQ ID No. 3.

Preferably, the peptide of the invention is chosen from one of the sequences as represented in formulas I and II below:

CRRWWRRWRR

RRR

aypqs ydHHVAWqdypcs

These peptides have better penetration properties.

The invention finally relates to a polynucleotide encoding the peptide of the invention. This polynucleotide is highly advantageous for use in gene therapy. Therapeutic applications of the peptide of the invention

The invention relates to the peptide as defined above for its use as a medicament. The invention also relates to the peptide as defined above for its use in the treatment of a motor neuronopathy selected from infantile spinal amyotrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease and Charcot's disease. Marie-Tooth disease. By "motor neurons" or "motor neurons" is meant the neurons constituting the exit path of the central nervous system (or the final pathway) of any motor act. The cellular bodies of motor neurons are located either in the brainstem or in the ventral horn of the gray matter of the spinal cord. Each motor neuron has an axon that starts from the central nervous system to innervate the muscle fibers of a muscle. The set consisting of a motor neuron and the muscle fibers it supplies is a motor unit. There are three types of motor neurons: the "alpha motor neurons", which innervate the muscle fibers responsible for contraction, the "gamma motor neurons" that innervate the neuromuscular spindles, thus adjusting their sensitivity to stretching, as well as the beta motor neurons. ", which innervate both types of fibers. Preferably, the compound of the invention is particularly advantageous for the treatment of motor neuronopathies involving the degeneration of alpha motor neurons.

By "motor neuronopathy" is meant a disease involving the degeneration of motor neurons, manifested by an absence of muscle stimulation leading to amyotrophy.

This disease is associated with a deficiency of SMN protein and / or a reduction in the number of CBs and / or a lack of localization of the SMN protein to CBs.

The symptoms of such a pathology may be varied and may include:

- a progressive motor deficit;

amyotrophy;

fasciculations; and or Cramps.

Said motor neuronopathy is infantile spinal amyotrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease, or Charcot-Marie-Tooth disease. Preferably, said motor neuronopathy is infantile spinal amyotrophy, amyotrophic lateral sclerosis. More preferably, said neuronopathy is infantile spinal muscular atrophy. Typically, said infant spinal muscular atrophy is type I, type II or type III.

The inventors have indeed made it clear that the peptide of the invention makes it possible to increase the distribution of the SMN protein to the Cajal bodies and / or to increase the number of Cajal bodies. Also, the peptide of the invention, by significantly increasing the distribution of SMN protein to Cajal bodies and / or the number of Cajal bodies, constitutes a relevant therapeutic strategy for the treatment of motor neuronopathies, in particular SMA. .

The peptide of the invention mimics the site of interaction between the proteins of the PP1 protein phosphatase family and Gemini 8. The family of PP1 protein phosphatases includes a very large number of holoenzymes which play a predominant role in many fundamental cellular functions such as apoptosis (Garcia et al., 2003, Godet et al., 2006), synaptic plasticity (Dehmelt and Halpain, 2007) and muscle contraction (Cohen, 2002). In this family of protein phosphatases, the catalytic subunits (C) are capable of associating with a very large variety of regulatory or so-called interacting subunits (Bollen et al., 2010). In mammals, there are three genes encoding the four catalytic subunits of PP1, PP1, PP1, β / δ and splice variants ΡΡΙγΙ (here, ΡΡΙγ) and ΡΡ1γ2, specific for the testis (Moorhead et al., 2007). ). The distribution, substrate specificity and activity of these catalytic subunits are defined by their association with the regulatory subunit (Bollen et al., 2010). In most cases, these proteins bind to the catalytic subunits PP1 by a degenerate R / Kx _(0jl) V / IxW / F consensus motif. Members of this protein-phosphatase family are also associated with pathological processes involved in diseases such as neurodegenerative diseases, heart failure, type 2 diabetes, cancer, and viral infections (Nuytten et al., 2008; Kelsall et al., 2009, Boyce et al., 2005, Nicolaou et al., 2009). Many inhibitors pharmacological targeting the catalytic subunits phosphatases PPl type were studied, their low selectivity vis-à-vis each of the holoenzymes, does not allow targeted use in human therapy. The inventors have succeeded in specifically targeting a given holoenzyme thanks to the development of the peptide of the invention. Indeed, the in vitro interaction observed between the immunopurified SMN complex and the recombinant protein ΡΡΙγ led them to search among the proteins of the complex for the signature of the PPl interaction motif. The potential motif HHVAW was found in the peptide sequence of Géminé 8. First of all, the inventors have demonstrated by in vitro binding studies that the Géminé 8 protein can interact directly with the γ -phosphatase protein. Moreover, the mutation of the HHVAW motif to HHAAA (SEQ ID No. 17) in re-localization experiments of the endogenous ΡΡΙγ protein with HeLa cells in culture demonstrates that it is involved in the recruitment of ΡΡΙγ by Géminé 8 to CBs. They have thus highlighted the fact that the Géminé 8 protein domain containing the HHVAW motif is essential for designing new peptide molecules with therapeutic potential against motor neuronopathies, in particular SMA.

The inventors have thus developed this new peptide, which is highly relevant for overcoming the deficit of SMN protein at the level of CBs. Indeed, the latter makes it possible to significantly increase the distribution of SMN in CBs.

Therefore, the peptide of the invention constitutes a therapeutic strategy that is relevant in the treatment of motor neuronopathies, in particular SMA or ALS.

By "Cajal body" or "Cajal bodies" or "CBs" we mean the site of the initial modifications and the assembly of several small nuclear RNAs with their ribonucleoproteins, the small nuclear ribonucleoproteins (SNRNPs) imported from the cytoplasm by the SMN complex, or recycled into the nucleus. The CBs have an ultra structure revealing coiled threads, hence their other name of "coiled bodies". CBs are dynamic structures and their number can change rapidly if there is a change in the level of transcription. The abundance of CBs can be easily determined by those skilled in the art, in particular by the immuno-detection of the coilin protein (CBs marker) and the number of CBs thus detected. As noted previously, SMN protein accumulates in Cajal bodies. A defect in this accumulation is described in the SMA.

Pharmaceutical compositions

The inventors have demonstrated a synergistic effect between the peptide of the invention and cyclosporin A for the distribution of SMN protein to CBs. This combination is therefore highly promising for the treatment of motor neuronopathies such as infantile spinal muscular atrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease or Charcot-Marie-Tooth disease.

Cyclosporin A or CsA is a cyclic peptide composed of eleven amino acids that binds to cyclophilins and causes specific inhibition of protein-phosphatase 2B (PP2B). Its IUPAC name is as follows: [R - [[R *, R * - (E)]] - cyclic (L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl- N-methyl-L-valyl-3-hydroxy-N, 4-dimethyl-L-2-amino-6-octenoyl-L-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl- N-methyl-L-leucyl). Its CAS number is 59865-13-3. Because of its immunosuppressive effect, CsA is already used in therapy in humans.

Cyclosporine is commercially available and is available from Cell Signaling.

The invention therefore also relates to a composition comprising:

a peptide comprising a penetration sequence (a) and a sequence (b) comprising the protein unit shown in SEQ ID NO: 1; and

cyclosporin A.

The technical characteristics developed above with respect to said peptide are applicable here. The inventors have shown that the peptide of the invention in combination with cyclosporin A synergistically exert a beneficial effect on the accumulation of SMN protein at CBs in fibroblasts of patients with the three forms of infantile spinal muscular atrophy. Therefore, the combination of the peptide of the invention with cyclosporin synergistically exerts a therapeutic effect.

The composition according to the invention may combine the peptide of the invention and cyclosporin A with other active agents, in particular active agents capable of treating motor neuronopathies. Among these active agents, there may be mentioned by way of example:

riluzole (Haddad H et al., Riluzole attenuates spinal muscular atrophy disease progression in a mouse model, 2003. 28 (4): 432-437 and Wadman RI et al., 2012. Drug treatment for spinal muscular atrophy type I, Cochrane Database Syst Rev. 4: CD006281);

hydroxyurea (Chen TH et al., Randomized, double-blind, placebo-controlled trial of hydroxyurea in spinal muscular atrophy, Neurology, 2010 75: 2190-21); ubiquitin / proteasome inhibitors (Chang HC et al., Degradation of motor neuron survival (SMN) protein is mediated via the ubiquitin / proteasome pathway, Neurochem Int., 2004, 45: 1107-1112 and Kwon DY et al. Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice, Hum Mol Genet., 2011, 20: 3667-3677);

histone deacetylase inhibitors (Lunke S et al., The Emergent Role of Epigenetic Modifications and Chromatin Remodeling in Spinal Muscular Atrophy, J. Neurochem, 2009, 109 (6): 1557-1569);

Protein phosphatase inhibitors (Novoyatleva T et al., Protein phosphatase 1 binds to the RNA recognition pattern of multiple splicing factors and regulates alternative pre-mRNA processing, Hum Mol., Genet., 2008, 17, 52-70);

Olesoxime (TR019622), Bordet T et al., Identification and characterization of cholest-4-en-3-one, oxime (TRO 19622), a novel drug candidate for lateral amyotrophic sclerosis, J Pharmacol Exp Ther, 2007, 322 (2): 709-720).

aminoglycosides (Mattis VB et al., Analysis of a read-through promoting compound in a severe mouse model of spinal muscular atrophy, 2012, 525 (1): 72-75);

salbutamol (Pane M et al., Daily salbutamol in young patients with SMA type II, Neuromuscul Disord., 2008, 18: 536-540);

the isoindoline (application US 2010/0267712);

ibudilast (US application 2009/0062330);

kinase modulators (Burnett BG, et al., Regulation of SMN protein stability, Mol Cell Biol., 2009, 29 (5): 1107-1115);

IGF-1 variants (Murdocca M et al., IPLEX improved motor neuron survival and improved motor functions in a severe mouse model of

SMA, Mol Med. 2012 May 29. doi: 10.2119 / molmed.2012.00056);

human growth factors (US application 2008/0187512);

bicyclic pyrimidines inhibiting PTK (Hastings ML et al., Tetracyclines that promote SMN2 exon 7 splicing as therapeutics for spinal muscular atrophy, Sci Transi Med., 2009, 1 (5): 5ral2);

EGFR inhibiting quinazoline (Butchbach ME et al., Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy, Hum Mol Genet., 2010, 19 (3): 454-467 ); tyrosine kinase inhibitors (application US 2010/0305036);

inhibitors of VEGFR (application US 2010/0331296);

HSP90 inhibitors (Suzuki K et al., Pathogenesis-targeting therapeutics for spinal and bulbar muscular atrophy (SBMA), Neuropathology, 2009, 29 (4): 509-516);

antagonists of myostatin (Rose FF Jr et al., Delivery of recombinant follicles in the disease model of spinal muscular atrophy,

Hum Mol Genet., 2009, 18 (6): 997-1005);

low molecular weight heparin (US application 2002/0040013);

- Neramexane (US application 2010/0234402);

nicergoline (application US 2003/0134869);

inhibitors of the G3SKB protein kinase (Makhortova NR et al., A screen for regulators of motor neuron protein levels, Nat Chem Biol., 2011, 7 (8): 544-552); RhoA and Rho-associated kinase inhibitors (ROCK pathway) (Bowerman M et al., Mol Cell Neurosci, 2009, 42: 66-74 and Bowerman M et al., Hum Mol Genet 2010, 19: 1468-1478);

antioxidants (Wan L et al., Inactivation of the SMN complex by oxidative stress, Mol Cell., 2008 31 (2): 244-254);

inhibitors of apoptosis (Sareen D et al., Inhibition of apoptosis blocks human motor neuron cell death in a stem cell model of spinal muscular atrophy, PLoS One 7 (6): e39113, 2012); or

modulators of the ERK and PI3K / AKT kinase pathways. (Biondi O et al., In vivo NMDA receptor activation accelerates motor unit maturation, protects spinal motor neurons, and enhances SMN2 gene expression in severe spinal muscular atrophy mice, J Neurosci., 2010 30: 11288-11299.)

Also, the invention also relates to a composition comprising at least one peptide according to the invention, at least one cyclosporin A and at least one active agent chosen from riluzole, hydroxyurea, ubiquitin / proteasome inhibitors, inhibitors histone deacetylases, protein phosphatase inhibitors, oleoxime, aminoglycosides, salbutamol, isoindoline, ibudilast, kinase modulators, IGF-1 variants, human growth factors, bicyclic pyrimidines inhibitors of PTK, EGFR-inhibiting quinazoline, tyrosine kinase inhibitors, VEGFR inhibitors, HSP90 inhibitors, myostatin antagonists, low molecular weight heparin, neramexane, nicergoline, inhibitors of G3SKB protein kinase, RhoA and Rho-associated kinase inhibitors (ROCK pathway), anti-oxidants, apoptosis inhibitors and modulators of ERK and PI3K / AKT kinases.

In the following, the expression "composition of the invention" designates a composition comprising:

a peptide comprising a penetration sequence (a) and a sequence (b) comprising the protein unit represented in SEQ ID NO: 1, and

a cyclosporin A,

with or without an active agent as defined in the two preceding paragraphs. Typically, the composition of the invention comprises Riluzole for use in the treatment of amyotrophic lateral sclerosis. The invention also relates to the composition of the invention for use as a medicament.

The invention also relates to the composition of the invention for use in treating a motor neuronopathy selected from infantile spinal amyotrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease and Charcot's disease. Marie-Tooth disease.

Preferably, the invention relates to the composition of the invention for its use in the treatment of infantile spinal muscular atrophy.

Typically, the combination of the peptide of the invention and cyclosporin can be simultaneous, separated or spread over time. Also, the invention relates to the peptide according to the invention and cyclosporin A for their simultaneous, separate or sequential administration for their use in the treatment of a motor neuronopathy chosen from infantile spinal muscular atrophy, amyotrophic lateral sclerosis, multiple sclerosis. primary syndrome, Kennedy's disease and Charcot-Marie-Tooth disease.

Finally, the invention also relates to pharmaceutical compositions for use in methods of treating motor neuronopathies in humans, said compositions comprising at least one peptide according to the invention and a pharmaceutically acceptable vehicle. Preferably, said pharmaceutical composition also comprises cyclosporin A.

The form of the pharmaceutical compositions, their route of administration, their dosage and their dosage naturally depend on the severity of the pathology, its stage of evolution, the age, the sex, the weight of the subject to be treated, etc. The skilled person so be sure to adjust the dosages according to the patient to be treated, especially during the treatment of SMA in which the patients are children.

The pharmaceutical compositions according to the invention can be formulated for topical, oral, systemic, intranasal, parenteral, intravenous, intramuscular, subcutaneous, or other administration. Depending on the mode of administration, the composition according to the invention may be in any galenic form.

Preferably, the peptide of the invention, and optionally cyclosporin are included in a composition that is administered orally. Orally, the compositions may be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid or polymeric vesicles allowing controlled release.

For topical application to the skin, the composition may have the form in particular of aqueous or oily solution or of dispersion of the lotion or serum type; emulsion of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O / W) or conversely (W / O); emulsion of soft consistency of the cream type; biphasic emulsion; aqueous or anhydrous gel; foam or microcapsules or microparticles, or vesicular dispersions of ionic and / or nonionic type, or sprayable formulations of "spray" type. These compositions are prepared according to the usual methods known to those skilled in the art. For systemic application, the composition may be in the form of an aqueous or saline solution.

Treatment method

The invention also relates to methods of treating a subject suffering from a motor neuronopathy, such as infantile spinal amyotrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease or Charcot-Marie disease. -Tooth including the step of administering to the subject a therapeutically effective amount of at least one peptide of the invention. By "therapeutically effective amount" is meant an amount sufficient to treat and / or stop progression motor neuronopathies.

The technical characteristics developed previously are applicable here.

All of the references cited in this application are incorporated by reference. FIG. 1: Quantitative Analysis of the Internalization of the DPT-5 Peptide in the Cells The HeLa cells (A) or the fibroblasts of SMA patients (B) were incubated for 6 hours with 125 nM peptides. In the presence of DPT-5 peptide, incorporation of 449 μg of HRP per 10 ⁵ HeLa cells and 519 μg of HRP per 10 ⁵ fibroblast cells of SMA patients was observed.

Figure 2: Effect of the different peptides on cell viability. The transformed human HeLa cells or fibroblasts were incubated for 24 hours in the presence of different concentrations of peptides. Survival was analyzed by the MTT test. Figure 3: Demonstration by immunostaining of the effect exerted on the proportion of HeLa cells for which the distribution of the SMN protein to Cajal bodies (CBs) is observed by treatment with peptide DPT-Sl according to its concentration in micromolar. A) histogram representing the dose-response of HeLa cells (chi-2 test, p <0.001, 300 <n <500 cells per condition) and

B) Confirmation of the effect exerted by the 25 micromolar peptide DPT-Sl with HeLa cells (chi-2 test, p <0.001, 6 experiments).

FIG. 4: Demonstration by immunostaining of the effect exerted on the immortalized fibroblasts of a patient suffering from the severe form of SMA for which the distribution of the SMN protein to the Cajal Bodies (CBs) is observed with the treatment with cyclosporin A following the concentration (nanomolar). Histogram representing the dose-response of the cells. 500 <n <1800 cells per condition; 2 (25 nM) or 3 independent experiments (10, 50 and 100 nM).

FIG. 5: Immunolabeling demonstration of the effect exerted on the immortalized fibroblasts of a severe form SMA patient (type I) for which the SMN protein distribution to the Cajal Bodies (CBs) is observed with the treatment with DPT-S1 synergistically with cyclosporin A (chi-2, p <0.001, 1300 <n <2200 cells per condition, 3 independent experiments). Figure 6. Demonstration by immunostaining of the effect exerted on fibroblasts in primary culture of a patient of each of the three forms of SMA for which the SMN protein distribution to Cajal bodies (CBs) is observed by DPT-S1 with cyclosporin A. The three forms of the disease range from type I (severe) to type III (moderate) disease. A value equal to 1 was given arbitrarily to the treatment with DPT-sh1 and CsA which serves as a control condition to calculate the induction value, (chi-2, p <0.001 500 <n <2200 cells per condition) .

Figure 7: Analysis of the internalization of HRP by peptides containing sequences from Géminé 8.

HeLa cells and fibroblasts of SMA patients are incubated for 6 hours with 125 nM streptavidin-HRP coupled biotinylated peptides. The negative control is obtained by incubating the cells with the same amount of streptavidin-HRP as that used with the peptides. 17.7 ± 8.2ng and 16.3 ± 5.3ng of HRP were internalized respectively in SMA fibroblasts and in HeLa cells by the TAT reference penetrant peptide which serves as a positive control. The data group together 3 independent experiments.

Figure 8: Biological effects of penetrating peptides derived from Gemini 8 protein on CBs with immortalized fibroblasts of the severe form of spinal muscular atrophy. The cells are treated for 16 hours. My data includes 7 independent experiments. Cells were treated with each of the peptides in the presence of cyclosporin A (10 nM). A clear increase is observed only with the DPT-S1 peptide which contains the Shl shuttle (Khi-2 test, p <0.001). EXAMPLES

1- Internai isation in the HeLa cell and the SMA fibroblast said new penetrating peptide DPT-S1 containing the Géminé 8 sequence. Method:

The immortalized HeLa cells or fibroblasts of an SMA patient with the severe form of type 1 (1 to ² × 10 ⁵ for 200 μl) are inoculated in 96-well plates with complete DMEM medium in the presence of 10% fetal calf serum. After overnight incubation at 37 ° C. in a CO 2 incubator (5%), the cells are incubated for 24 hours at 37 ° C. in the presence of 125 nM of biotinylated peptides, which have themselves been preincubated for 40 minutes at room temperature. with Streptavidin-horseradish peroxidase (HRP) in a proportion of 4 moles of peptides per 1 mole of Streptavidin-HRP. After 6 hours of incubation, the cells subjected to a trypsinization step, then are washed with PBS and centrifuged for 10 min at 600 g. The cell pellet is taken up with 0.3 ml of lysis buffer (0.1 M Tris-HCl pH 8, 0.5% NP-40 buffer) and left for 10 minutes on ice. After centrifugation for 10 minutes at 4 ° C. and 13000 g, the cell debris is removed and then the lysis supernatant is recovered, which is then incubated with 50 μl of OPD buffer (25.7 ml 0.2 M dibasic sodium phosphate + 24.3 ml). 0.1 M citric acid + 50 ml of distilled water, adjusted to pH 5.0) and 50 μl of OPD solution (one OPD tablet "Free base of SIGMA CHEMICAL COMPANY P-5412" is dissolved in 50 ml of buffer OPD with 40 μΐ H ₂ 0 ₂ 30%). The reaction, about 15 minutes, is stopped by adding 100 μΐ 1M HC1. The peroxidase activity is determined by reading at 490 nm to the ELISA reader. The amount of peroxidase in the lysates is calculated from the reference curve and then referred to the same number of cells. Results:

The inventors analyzed, in the HeLa cells and in the SMA fibroblasts, the internalization of the protein-Streptavidin-HRP complex by the peptide DPT-S1 containing the sequence derived from Géminé 8 (residues 77-88, SEQ ID No. 1). associated with the sequence of the shuttle DPT-shl (SEQ ID No. 2).

Figure 1 shows that, as previously described by the inventors, the DPT-shl shuttle alone does not internalize HRP complex.

The addition of the sequence 77-88 of Géminé 8 (SEQ ID No. 1), containing a potential interaction pattern of Géminé 8 with ΡΡΙγ, demonstrates the "cargo" property and demonstrates the penetrating character of the bipartite DPT peptide. -S1. 2- Effect of the Penetrating Peptide Containing a Géminé 8-derived Sequence on the Viability of HeLa Cells and SMA Patient Fibroblasts

Method:

The Sigma MTT-Kit protocol (ref G4000) was followed for this experiment.

HeLa cells or SMA fibroblasts (3000 cells per 200 μl) are seeded in 96-well plates with complete DMEM medium in the presence of 10% fetal calf serum. After overnight incubation at 37 ° C. in a 5% CO ₂ oven, the cells are cultured in the presence of different concentrations of peptides added directly to the culture medium. After 24 hours of incubation, 10 μl of the MTT solution of the kit per well are added. The incubation is carried out in the dark at 37 ° C. for 3 hours before adding 100 μl of the "Solubilization / STOP" solution of the kit per well. After homogenization of the wells with the pipette, the plates are incubated for 1 h at 37 ° C. which allows complete lysis of the cells. This makes it possible to homogenize the dissolution of the reaction product in the wells before reading the plates at 570 nm with a reference filter at 630 nm. Results:

The inventors analyzed the toxicity of peptides DPT-sh1 and DPT-1s after 24 hours, incubating with both HeLa cells and SMA fibroblasts. The results indicate that DPT-Sl used between 1 and 50 μΜ has no significant toxic effect on HeLa cells (Fig.2A) and on SMA fibroblasts (Fig.2B).

3- Effects exerted on the distribution of the SMN protein at the CBs with the HeLa cells and the SMA fibroblasts during the treatment with the peptide DPT-Sl

Method & Results

The first series of experiments was first used to validate on HeLa cells in culture the effects exerted on the CBs by the peptide DPT-Sl. To evaluate the relationship between the concentration (dose equal to 5, 10, 25 and 50 micromolar) and the effect (response) on the CBs of the HeLa cells, we first constructed a dose-response curve with the DPT peptide. IF comparing it to the DPT-shl shuttle alone (Fig. 3).

The increase in the number of CBs in HeLa cells (10,000 cells per well of 0.8 cm) after 16 hours of treatment was analyzed for each concentration. To determine whether the DPT-Sl peptide exerted a positive effect, we counted the proportion of cells that had SMN protein at 0 or 2 CBs, at 3 or 4 CBs and at 5 CBs and above, in randomly selected cells. every experience.

Immunostaining analysis using the specific antibodies (commercial and custom-produced) of the SMN and coilin proteins (another marker of the CBs) shows that the shuttle alone has no effect on the location of the SMN protein at the same time. CBs (Fig. 3A).

The proportion of HeLa cells with an increase in the number of CBs (3 to 4) increases with the increase in the concentration of DPT-Sl peptide. The results obtained from this dose-response experiment show that 25 micromolar DPT-5 significantly increases the number of CBs (chi-2, p <0.001, total number of cells counted for each condition is at least 300).

We then repeated the 25 micromolar treatment (Fig. 3B); and data obtained from six independent experiments concluded that this peptide was able to significantly increase the distribution of SMN protein to CBs in HeLa cells (chi-2, p <0.001, n = 1320 cells for DPT-shl and n = 2163 cells for DPT-S1).

In search of the molecular processes involved and inducing the effects on CBs, we have evaluated the role that protein-phosphatase 2B (PP2B) could play.

The inventors have decided to test cyclosporin A. It is a cyclic peptide composed of eleven amino acids which binds to cyclophilins and causes specific inhibition of PP2B. With its immunosuppressive effect, CsA is already used in therapy in humans.

The inventors first evaluated the relationship between the concentration (10, 25, 50 and 100 nanomolar) and the beneficial effect on CBs of immortalized SMA fibroblasts (Fig. 4). The median effective concentration (EC50) of CsA in our experimental conditions is about 25 nanomolar. On the other hand, it has been reported that the EC50 value for CsA was 18 nM in a study on the nucleocytoplasmic translocation of nuclear factor NFATcl, substrate of PP2B (data sheet NFATcl redistribution assay, Thermo Scientific) . The value found here is therefore compatible with the concentrations associated with the effects of CsA specifically targeting the activity of PP2B protein phosphatase.

The inventors have made the hypothesis that the DPT-S1 peptide (at 25 micromolar) and the CsA (at 10 nanomolar) could act synergistically on the distribution of SMN protein to CBs in patient fibroblasts. At this concentration, the inventors have previously shown that CsA has no detectable effect (FIG 4). The inventors then conducted similar immunostaining experiments to the previous ones using SMA fibroblasts. immortalized treated or not with a combination of DPT-Sl peptide and CsA (Fig. 5).

The results obtained from 3 independent experiments made it possible to conclude the synergistic effect between the DPT-Sl and the CsA (chi-2, p <0.001, 1300 <n <2200 cells per conditions). We also investigated the effect after 16 hours of treatment with DPT-Sl and CsA on increasing the number of CBs with SMN in primary cultures of type I (severe), type II (intermediate form) fibroblasts. and type III (moderate form). The synergistic action of peptide and CsA on CBs was also found with primary fibroblast cultures (Fig. 6). The cultures of the three forms globally respond in the same way with an induction factor of a mean value equal to about 2. This similarity could be linked to the existence of a feedback loop already reported with the SMN protein. Statistical analysis from these experiments has shown that the DPT-Sl peptide with CsA synergistically exerts a beneficial effect on the accumulation of SMN protein at CBs in fibroblasts of patients with all three forms of the disease. . Conclusion

The inventors have thus demonstrated that a new peptidic agent DPT-Sl has a promising beneficial effect because it allows recruitment of SMN protein to CBs in HeLa cells and fibroblasts derived from the skin of children with motor neuronopathy. especially infantile spinal amyotophy. 4. Analysis of the influence of the penetration sequence

The inventors have developed several peptides to study the influence of the penetration sequence on, inter alia, the effectiveness of the peptide to produce and-accumulation of the SMN protein to CBs in fibroblasts. The sequences of the peptides synthesized are summarized in the table below:

DPT-S2 * contains binding pattern from Bcl2 to PP1 alpha

DPT-Sl and DPT-S3 contain the wild binding pattern and are differentiated by the shuttle used

In the context of the following experiments, each of the sequences is grafted to a biotin.

A. Analysis of the Internalization of HRP by Peptides Containing Sequences Derived from Gemini 8

HeLa cells and SMA patient fibroblasts are incubated for 6 hours with 125 nM streptavidin-HRP coupled biotinylated peptides. The negative control is obtained by incubating the cells with the same amount of streptavidin-HRP as that used with the peptides.

17.7 ± 8.2ng and 16.3 ± 5.3ng of HRP were internalized respectively in the SMA fibroblasts and in the HeLa cells by the TAT reference penetrant peptide which serves as a positive control (FIG. 7). These results indicate that the TAT shuttle that allows penetration does not have an effective biological effect.

Also, the DPT-S4 sequence is not sufficient to induce the biological effect resulting from the specific interaction of ΡΡΙγ with DPT-S4. This clearly indicates that the intracellular localization of DPT-Sl, distinct from those of TAT & DPT-S3, is important for the biological effect.

Therefore, these results show that the nature of the penetration sequence influences the effectiveness of the peptide.

B. Comparison of the biological effects of peptides

HeLa cells are treated for 16 hours with each of the peptides and double-labeled immunofluorescence experiments are performed. The CBs are detected using 2 specific antibodies, one for the coilin protein and the other for the SMN protein. The presence of the SMN protein at the CBs is revealed during a co-localization of the 2 antibodies to the punctiform structures in the nucleus of the cells (n> 6).

The optimal number of CBs was shown to be 3 or 4 per nucleus with HeLa cells (Stanek et al, 2008).

Here, the treatments with peptides DPT-Sl and DPT-S3 show a significant increase in the number of HeLa cells having 3 or 4 CBs (Khi-2, p <0.001). The results show that the induction effect is positively superior with the DPT-Sl peptide (containing the Sh1 shuttle) than with the DPT-S3 peptide (containing the TAT shuttle).

C. Conclusion

Regarding the penetration and intracellular localization of DPT-Sl, the replacement of the DPT-shl shuttle by the Tat shuttle demonstrates that penetrating the DPT-S4 sequence is not sufficient to induce the biological effect resulting from the specific interaction of ΡΡΙγ with DPT-S4. This clearly suggests that the intracellular localization of DPT-Sl, distinct from those of TAT & DPT-S3, is important for the biological effect. Concerning the specific interaction of ΡΡΙγ with DPT-Sl, we have shown that DPT-S2, the inactive mutant peptide of DPT-Sl in which the interaction pattern of ΡΡΙγ has been replaced by that of PPla, enters the cell via interaction with PPla without any influence on the number of CBs.

These results also show that the peptide DPT-S 1 exerts a biological effect in combination with cyclosporin A, this effect is not observed with peptide DPT-S3 (FIG. 8).

Finally, these results confirm that the peptide penetration sequence can not be chosen arbitrarily, and that the specific selection of this peptide confers a biological effect, essential for its effective use in therapy.

5. Analysis of the Gemini-Derived Peptide Efficacy Having a Disulfide Point Penetration Sequence The inventors have developed peptides having a penetration sequence having a disulfide bridge between two cysteines.

The sequence (a) of penetration of the peptides is as follows:

the amino acid sequence represented in SEQ ID No. 28 (CRRRRRRRRRR), whose cysteine in position 1 forms a disulfide bridge with the cysteine at position 18 of the sequence SEQ ID No. 3. The peptides thus obtained are as follows: CRRWWRRWRR

aypqsfydHHVAWqdypcs

CRRRRRRRRR

(■■)

S I aypqsfydHHVAWqdypcs

HeLa cells are treated for 16 hours with each of the peptides at different concentrations and double-labeled immunofluorescence experiments are performed. The CBs are detected using 2 specific antibodies, one for the coilin protein and the other for the SMN protein.

The presence of the SMN protein at the CBs is revealed during a co-localization of the 2 antibodies to the punctiform structures in the nucleus of the cells (n = 3).

The results show an improvement in the penetration properties of these peptides.

Claims

claims

1. Peptide comprising:

a biologically inactive (a) penetration sequence;

a sequence (b) comprising the protein unit represented in SEQ ID No. 1.

Peptide according to claim 1, characterized in that said penetration sequence (a) is a cationic amino acid sequence comprising between 6 and 8 amino acids chosen from arginine and lysine residues.

Peptide according to claim 1 or 2, characterized in that said penetration sequence (a) is selected from the group consisting of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO. ID N ° 16.

Peptide according to claim 1 or 2, characterized in that said penetration sequence (a) is chosen from:

the amino acid sequence shown in SEQ ID NO: 27, whose cysteine at position 1 forms a disulfide bridge with cysteine at position 18 of the sequence SEQ ID NO: 3; and

the amino acid sequence shown in SEQ ID NO: 28, whose cysteine in position 1 forms a disulfide bridge with the cysteine at position 18 of the sequence SEQ ID No. 3.

Peptide according to claim 1 or 2, characterized in that said penetration sequence (a) has an amino acid sequence as represented by SEQ ID NO: 2.

6. Peptide according to any one of claims 1 to 5, characterized in that said sequence (b) has an amino acid sequence as represented by SEQ ID No. 3.

7. Peptide according to any one of claims 1 to 5, characterized in that said peptide consists of the sequence represented by SEQ ID No. 4 or in a sequence having at least 80% identity with the sequence SEQ ID N # 4.

Peptide according to any one of claims 1 to 7 for use as a medicament.

A peptide according to any one of claims 1 to 7 for use in the treatment of a motor neuronopathy selected from infantile spinal amyotrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease and Parkinson's disease. Charcot-Marie-Tooth.

Peptide for use according to claim 9, characterized in that said motor neuronopathy is infantile spinal muscular atrophy.

Peptide for use according to claim 9 or 10, characterized in that it allows the increase of the distribution of the SMN protein to the Cajal bodies and / or the increase in the number of Cajal bodies.

12. Composition comprising:

a peptide according to any one of claims 1 to 7, and

cyclosporin A.

13. Composition according to claim 12, further comprising an active agent chosen from riluzole, hydroxyurea, ubiquitin / proteasome inhibitors, histone deacetylase inhibitors, protein phosphatase inhibitors, oleoxime, aminoglycosides, salbutamol, isoindoline, ibudilast, kinase modulators, IGF-1 variants, human growth factors, bicyclic pyrimidines inhibiting PTK, EGFR-inhibiting quinazoline, tyrosine kinase inhibitors, inhibitors of VEGFR, HSP90 inhibitors, myostatin antagonists, low molecular weight heparin, neramexane, nicergoline, G3SKB protein kinase inhibitors, RhoA inhibitors and of Rho-associated kinase (ROCK pathway), anti-oxidants, apoptosis inhibitors and modulators of ERK and PI3K / AKT kinases

14. The composition of claim 12 or 13 for use as a medicament.

A composition according to claim 12 or 13 for use in the treatment of a motor neuronopathy selected from infantile spinal muscular atrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, Kennedy's disease and Charcot-Marie-Claude's disease. Tooth.

16. Composition according to claim 12 or 13 for its use in the treatment of infantile spinal muscular atrophy.

Peptide according to any one of claims 1 to 7 and cyclosporin A for their simultaneous, separate or sequential administration for use in the treatment of a motor neuronopathy selected from infantile spinal muscular atrophy, amyotrophic lateral sclerosis, multiple sclerosis. primary syndrome, Kennedy's disease and Charcot-Marie-Tooth disease.